The role of dendritic cells in the pathogenesis of leishmaniasis

In humans, leishmaniasis comprises a spectrum of disease from cutaneous lesions to destructive mucocutaneous erosions or an insidiously progressive fatal visceralising disease. An estimated 1 to 1.5 million people are afflicted each year with cutaneous leishmaniasis and 500 000 victims succumb to visceral disease per annum worldwide. Leishmaniasis is caused by protozoan parasites, Leishmania, which are transmitted by the phlebotomine sand fly to the host where they become obligate intracellular parasites of mononuclear phagocytes. The protozoa are dimorphic. The flagellated promastigote (PM) form of the parasite from the sandfly inoculum enters the host cell where it transforms into a compact amastigote (AM) without flagellum in a parasitophorous vacuole (PV) within 24 hours and replicates by binary fission. Whether infection remains asymptomatic or develops into locally aggressive or disseminated disease is based in part on parasite type and virulence factors but predominantly on the host immune response. Cell mediated immunity is required for control of infection following transmission of the parasite. This is dependent upon the type of interaction which occurs between the antigen presenting cells (APC), the first host cells to encounter the parasite and T helper (Th) cells. Parallels with human leishmaniasis can be drawn from the murine model of Leishmania major infection. In genetically inbred mice there is distinct Th cell differentiation, characterised by their cytokine profile, which correlate with susceptibility or resistance to disease. The events which direct Th differentiation in susceptible and resistant mouse strains remain controversial but are believed to operate in the first hours or days of infection. There is a body of evidence to suggest that dendritic cells (DC), the most potent APC, play a role in leishmaniasis. However their interaction had only been investigated with AM, not the initial infectious PM stage. Whether DC interact with PM and if so, whether this interaction plays a significant role in the primary immune response to Leishmania, particularly in Th differentiation, is a tantalising but as yet unanswered question. Two studies are described in this thesis. The major study involved original laboratory research investigating the role of dendritic cells in the pathogenesis of leishmaniasis. The supplementary study was a clinical field study undertaken in an hyperendemic area for cutaneous leishmaniasis (CL) in Syria to trial a novel topical therapy for treatment of CL. The major study was based on the development of a murine in-vitro model to investigate the hypothesis that DC internalise L.major PM and play a pivotal role in the early events that shape the development of a cellular immune response to infection. Conditions were defined for optimal DC internalisation of L.major PM and their stimulation of naive T cell responses to leishmanial antigens. Their potential to influence Th development, by cytolcine production or modulation of costimulator expression, was then investigated. The original findings from this research suggest that DC play an important role in establishing an early primary T cell response to L.major PM. DC internalise L.major PM with maximal expression of co-stimulatory and MHC Class II molecules and in response produce IL-12. Combined with results of the primary stimulation assays these findings suggest that DC from both susceptible and resistant mouse strains are able to stimulate autologous naive T cells when pulsed with PM antigens and provide a cytokine environment predicted to favour the development of a Th 1 response. The importance of DC in the overall immune response to Leishmania is further underlined by observations that macrophages appear to be temporarily paralysed during infection with L.major PM, particularly in their production of IL-12, due to a number of evasion strategies which the parasite has evolved. This work also identifies a potential use of DC in the search of non-peptide antigens shed by L. major which are presented via non-classical MHC pathways, as potential vaccine candidates. The supplementary study involved the development and clinical study of a nitric oxide (NO) generating cream in the topical treatment of CL. CL often has a protracted period of healing, for months and occasionally years, with potential for secondary bacterial infection and significant disfigurement. Current first line treatment entails repeated intralesional injections which are painful, time consuming, expensive and now associated with increasing parasite resistance. Improvement in treatment is greatly needed. Nitric oxide synthesised by macrophages is lethal to Leishmania. Since NO diffuses into tissues, we reasoned that NO-generating creams applied topically to lesions might be an effective and inexpensive treatment for CL. The theory that NO can be generated non-enzymatically by the acidification of nitrite (KNO2) by a weak acid such as ascorbic acid (ASC), potassium chloride (KCL) or salicylic acid (SAL). Initial laboratory experiments performed by colleagues demonstrated efficacy of this topical combination therapy in vitro and in mice. We designed the clinical study to trial the nitrite cream in combination with one of the three weak acids for four weeks in 40 patients with CL in Aleppo, Syria, and assess clinical responses. Only 11(28%) of 40 patients showed improvement and only 5 (12%) of 40 were cured at 2 months. The investigation was small but demonstrated reasonably good patient tolerability and ease of application. Given the results of this study, and the urgent need for new, more easily applicable therapy, further development of NO-generating creams is warranted with assessment in placebo-controlled randomised trials. Both studies have been published in good peer-reviewed journals advancing scientific knowledge in both pathogenesis and treatment of leishmaniasis. Both areas are inevitably linked and advances made in the former will hasten the development and refinement of much needed treatments and ultimately, prevention of disease.